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Culture of Cancer Cell Lines: The authenticated (RADIL, Columbia, MO), human pancreatic cancer cell lines BXPC-3 (witout ras mutations) and Panc-1 (expresses activating point mutations in K-ras) originally purchased from the American Type Culture Collection (Rockville, MD) were grown in RPMI-1640 with 10% fetal bovine serum without antibiotics. A) Determination of cell proliferation by MTT assay: The colorimetric 3-(4, 5-dimethyle thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay (Sigma) was used as previously described [38] to assess the growth stimulating effects of chronic epinephrine exposure and potential inhibitory effects of celecoxib on this response in Panc-1 and BXPC-3 cells in vitro. Briefly, Panc-1 and BxPC-3 cells were seeded into 6-well plates (50,000 cells per well). Cells were then pretreated with 15 nM epinephrine for 7 days (medium containing epinephrine was replaced every 24 hours) or cultured for 7 days without epinephrine. Celecoxib (1 nM to 100 µM) was then added to cells from both treatment groups. Following a 72 hour incubation period, all cells were harvested.
B) Assessment of cell migration by colorimetric assay: Cell migration assays were conducted as previously described [36], using 6-well plates with filter inserts provided by the colorimetric cell migration assay kit (Cell Biolabs, San Diego, CA, USA). Panc-1 and BxPC-3 cells were pretreated with 15 nM epinephrine for 7 days (medium containing epinephrine was replaced every 24 hours) in tissue culture flasks or cultured without epinephrine for 7 days. Cells from both treatment groups were then seeded into the inserts and treated with celecoxib (1 nM to 100 µM) for 24 hours. The migratory ability of cells was then assessed following the vendor’s instructions.
C) Statistical evaluation of in vitro data: Data (n = 5) in column graphs (Fig. 1) from the cell proliferation and cell migration assays in the presence and absence of chronic epinephrine treatment were assessed by one-way analysis of variance followed by Dunn’s multiple comparison test. Data (n = 5) from the dose-response experiments (Fig. 2) with celecoxib were fitted to sigmoidal dose-response curves and EC50 values for celecoxib were calculated by nonlinear regression analysis using Prism GraphPad software.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0043376.g001 Results of MTT and migration assays.MTT assays (A) in Panc-1 (with activating point mutations in K-ras) and BXPC-3 (without ras mutations) cells in vitro. Exposure for 7 days to epinephrine (15 nM) significantly (p<0.001) increased the number of viable cell in both cell lines. The Cox-2 inhibitor celecoxib (1 µM) completely blocked this response to epinephrine (p<0.001) while additionally reducing (p<0.001) the number of viable cells in cells not pre-treated with epinephrine. The effects of identical treatments on cell migration are shown in Fig. B. In both cell lines migration was significantly more stimulated by chronic epinephrine than cell proliferation. Celecoxib significantly (p<0.001) reduced epinephrine-induced migration but did not completely block this response. BCPC-3 cells were slightly more responsive to epinephrine in both assays, but the differences between the two cell lines were not significant. Columns are mean values and standard deviations of 5 samples per treatment group.
Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0043376.g002 Cell proliferation and migration dose response-curves.Dose response-curves for celecoxib in Panc-1 and BXPC-3 cells in vitro in the presence and absence of pretreatment for 7 days with epinephrine (15 nM). Figures A and B show cell proliferation responses in MTT assays while Figures C and D show cell migration responses. While both cellular responses were similar among the two cell lines, EC50 values for celecoxib in Figs. B, C and D were significantly (p<0.001) lower in the cells pretreated for 7 days with epinephrine. Data points are mean values and standard deviations (n = 5). EC50 values and curve fit were established by nonlinear regression analysis for sigmoidal dose-responses.
Six-week-old male athymic nude mice were purchased from Harlan Sprague Dawley. The animal research protocol was approved by the University of Tennessee Institutional Animal Care and Use Committee. The mice were acclimated for 1 week and maintained in our laboratory animal facility in accordance with guidelines of the American Association of Laboratory Animal Care under standard laboratory conditions in which temperature, humidity, and light are controlled, and had free access to autoclaved Purina Rodent Chow food and autoclaved water. Following the 1-week acclimation, the ears of the mice were tagged to facilitate the monitoring of each animal, which was then randomly assigned to six treatment groups (n = 20), with 5 mice per cage. Before tumor cell inoculation, mice in the three psychological stress groups were exposed to social stress for 4 weeks according to the published procedure [37] by changing the group composition of each cage twice a week. BXPC-3 cells that had reached 75% confluency in culture were then subcutaneously inoculated in the flank region (3×106 in 0.2 ml of PBS, viability >95%) of animals from each of these groups. Social stress was continued in these three groups for another 30 days. Three other groups of mice that were not exposed to social stress were also inoculated with identical numbers of pancreatic cancer BXPC-3 cells. One group each from the social stress and non-social stress populations was treated by intraperitoneal injections of celecoxib (Celebrex, Pfizer; 25 mg/Kg b.w., 5 days/week for 30 days). Additionally, one group each from the social stress and non-social stress populations was simultaneously treated by initial intraperitoneal injections of celecoxib (25 mg/Kg b.w., 5 days/week for 30 days) and then GABA (Sigma; 10 mg/Kg b.w., 5 days/week for 30 days) immediately thereafter on the mouse’s opposite side. All animals were observed for 30 days after inoculation with cancer cells. Tumor sizes were evaluated weekly by digital caliper, and two perpendicular diameters (length and width) of each xenograft were measured using the following formula: tumor volume = (length/2)×(width2). The weight of the animals was followed throughout the experiment to monitor their general health state and for GABA and celecoxib treatment effects. At the end of the 30-day observation period, the animals were euthanized by CO2 inhalation. Blood samples were collected for the determination of neurotransmitters, vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE2) in the serum and of cAMP in the cellular fraction of blood. The tumors were excised, snap frozen in liquid nitrogen, and then stored at −80°C for further analyses.
B) Immunoassays for the quantitative analysis of the second messenger cAMP, VEGF, PGE2, and phosphorylated signaling proteins: VEGF and PGE2 in serum and tumor tissue samples and cAMP in the cellular fraction of blood and tumor tissues samples were analyzed by immunoassays as recommended by the manufacturer (Enzo Life Sciences International). Briefly, the kit for measurement of cAMP uses a polyclonal antibody to bind a competitive manner the cAMP in the standards and samples. The kit for measurement of VEGF uses monoclonal antibody to human VEGF immobilized in a microtiter plate to bind the VEGF in the standards or samples. The PGE2 kit uses a monoclonal antibody to PGE2 to bind in a competitive manner the PGE2 in the samples and standards. Absorbances were read with an ELISA reader at 405 nm for cAMP, 450 nm for PGE2, and at 450 nm for VEGF (n = 5 per treatment group). Detection and quantitation of levels of p-ERK-1/2 at threonine 202 and tyrosine 204, p-CREB at serine at 133, and p-Akt at serine residue 473 (Invitrogen); and kinase activity of the recombinant catalytic domain of p-Src (MBL International) were conducted from tumor tissue samples (n = 5 per treatment group) homogenized in RIPA buffer and protease inhibitors as recommended by the manufacturers. Absorbance was read with an ELISA reader at 450 nm. Serum and tumor levels of the stress neurotransmitters noradrenaline and adrenaline as well as GABA were determined by ELISA assays as previously described [14], [18] to monitor the successful induction of psychological stress (data not shown).
C) Semi-quantitative analysis of COX-2, 5-lipooxygenase (5-LOX) and p-5-LOX, and non-quantitative visualization of signaling proteins by western blotting: Three independent Western blots were conducted for each antibody for the semi-quantitative assessment of protein expression by densitometry for the AA-metabolizing enzymes COX-2 and p-5-LOX, using NIH Image J software. In addition, protein expression of the signaling molecules quantitated by ELISA assays above were visualized by non-quantitative Western blots. Briefly, tumor tissues were briefly homogenized in RIPA lysis buffer (Thermo Scientific), PMSF, Na3VO4, and 1 mg/ml aprotinin, leupeptin, and pepstatin. After heat denaturation at 100°C for 5 min, equal amounts of protein were electrophoresed using 12% Novex SDS-polyacrylamide gels (Invitrogen) and transferred onto nitrocellulose membranes; Western blots were then performed using incubation overnight at 4°C with the following primary antibodies: total ERK1/2, p-ERK1/2, p-CREB, AKT p-AKT, Src, p-Src, COX-2, 5-LOX, and p-5-LOX (Cell Signaling). Total CREB and actin were purchased from Millipore. Secondary antibodies were incubated for 1 h at room temperature. Bands were visualized by ECL (Pierce, Thermo Scientific).
D) Statistical analysis of in vivo data: Statistical analysis was performed using GraphPad Instat software (GraphPad Software Inc., La Jolla, CA, USA). To test if the variation among column medians of tumor volumes in the six treatment groups (n = 20) was significantly greater than expected by chance, nonparametric Kruskal-Wallis ANOVA was performed for data from each of the 4 weeks after injection of tumor cells. Differences between selected pairs of treatment groups were additionally assessed by the nonparametric Mann-Whitney test. Statistical significance of differences among groups for levels of cAMP (n = 5), PGE2 (n = 5), and VEGF (n = 5) in blood and xenograft tissues and for p-ERK, p-CREB, p-Src, and p-AKT in tumor tissue (n = 5 per treatment group) was assessed by the non-parametric Mann-Whitney test. Statistical significance of differences between four densitometric readings per protein band from three independent Western blots for the semi-quantitative assessment of COX-2 and p-5-LOX prepared from three randomly selected xenografts per treatment group (n = 12) was assessed by the non-parametric Mann-Whitney test.
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